The objective of this proposal is to characterize the receptors, signaling pathways and cellular mechanisms involved in relaxation of gastric smooth muscle by peptide (VIP, PACAP) and non-peptide (NO) neurotransmitters of the myenteric plexus. Several advances made during the previous funding period form the basis of the proposal. We have identified two receptor types for which VIP and PACAP have high affinity: a cognate, seven-transmembrane VIP2/PACAP3 receptor coupled via GS to adenylyl cyclase, and a distinct, single-transmembrane natriuretic peptide clearance receptor (NPR-C) coupled via Gi1 and Gi2 to activation of a constitutive NO synthase (NOS) in smooth muscle cells. We have identified the NOS isoform as eNOS by RT-PCR in muscle cultures and by in situ RT-PCR in single muscle cells. The concurrent stimulation of cAMP and cGMP has important implications with respect to their regulation by specific phosphodiesterases (PDE), activation and cross-activation of cA-kinase and cG-kinase, and feedback regulation of adenylyl cyclase. Preliminary studies have identified the isoforms of PDE (PDE3, PDE4, PDE5) and adenylyl cyclase (types V and VI) expressed in gastric muscle. Accordingly, the first aim is to characterize NPR-C and VIP2/PACAP3 receptors: the studies involve experiments in eNOS minus/minus and NPR-C minus/minus mice, reconstitution studies in COS-1 cells co-transfected with NPR-C and eNOS, site-directed mutagenesis to identify binding and G protein coupling domains, and chimeric constructs and site-directed mutagenesis to identify VIP2/PACAP3 receptor subtypes. The second aim is to characterize the regulation of adenylyl cyclases V/VI by cA-kinase, G proteins and Ca2+ influx; and the regulation of cAMP levels by cAMP-specific PDE4 and PDE3 and cGMP levels by cGMP- specific PDE5. The third aim is to characterize the concurrent activation and cross-activation of cA- and cG-kinase, and their role in phosphorylation of major targets in smooth muscle, including PLC-beta and PLA2, IP3 and ryanodine receptors, and MLC phosphatase. The combined approach embodied by these aims, all of which are supported by preliminary studies, should advance our knowledge of the cellular mechanisms mediating visceral smooth muscle relaxation.